TANNINS

1. TANNINS

2. Tannins: Introduction Tannins: pale yellow to light brown-red amorphous substances widely distributed in plants and used chiefly in tanning leather, dyeing fabric, and making ink. Their solutions are acid and have an astringent taste. They are isolated from oak bark, sumac, and galls. Tannins give tea astringency, colour, and flavour. Tannins are used industrially to clarify wine and beer, reduce viscosity of oil-well drilling mud, and they have also had medical uses Tannins are mainly found in the cell vacuoles or in the cuticle (here cells don’t interfere with plant processes: only after death and cell breakdown do they become active in metabolic processes). Tannins are phenolic glycosides.

3. TANNINS • Historically, the importance of tannin-containing drugs is linked to their tanning properties (ability to transform fresh hides into leather). • Today, tanning is achieved with mineral compounds, but for several millennia, it had required exclusively the used of plant products. • These included chestnut tree tannin (Castanea sativa L.), oak tannin (Quercus robar L) in Europe, but also, in other parts of the world, tannins from Anacardiacea (Quebracho – Schinopsis spp), sumacs (Rhus spp), Legumes (acacias – Acacia spp) or Combretaceae (myrobalans – Terminalia spp). • Some of these tannins are still prized for leathers designed for specific uses (fine leather products).

4. Tannins - Tanning The consequence of tanning: formation of bonds between the collagen fibres of the hide (imparts resistance to water, heat and abrasion.) This capability of tannins to combine with macromolecules explains why they precipitate cellulose, pectins and proteins; it also explains their characteristic astringency and tartness: by precipitating the glycoproteins contained in saliva, tannins make the latter lose its lubricating power.

5. The combination between tannins and macromolecules is established by hydrophobic interactions and hydrogen bonds between the phenolic groups of tannins and the proteins or other polymers. • Other types of linkages (irreversible) must also be involved to ensure the lasting stability of the combination between tannins and collagen structures. • These include the covalent bonds established after oxidation of the phenols to quinones. • The condition necessary for the formation of these linkages is the tannin’s molecular weight must fall within a well defined range. If it is too high, the molecule cannot insert itself into the interfibrillar spaces of the macromolecule. If it is too low, the molecule can insert itself but cannot form enough bonds to stabilize the combination.

6. Morocco – Tanning Pits

7. Tannins - Definition • “water-soluble phenolics of molecular weight between 500 and 3 000, which, in addition to displaying the classic reactions of phenols, can precipitate alkaloids, gelatine, and other proteins.” • A more recent definition of tannins: “Phenolic natural products that precipitate proteins from their aqueous solutions”.

8. True tannins: Molecular weights of 1000-5000. Pseudo-tannins: Gallic acid, catechins and chlorogenic acid are similar phenolic compounds: have a low molecular weight. True Tannins Vs Pseudo-Tannins

9. Tannins: Microscopy

10. Classification of Tannins

11. Classification of Tannins • In higher plants, two groups of tannins are generally distinguished, which differ by their structure, as well as their biogenic origin – hydrolysable tannins and condensed tannins. • Hydrolysable Tannins: Ellagitannins Gallotannins

12. Hydrolysable Tannins • Hydrolyzable tannins are hydrolyzed by weak acids or weak bases to produce carbohydrate and phenolic acids. • Examples of gallotannins are the gallicacid esters of glucose in tannic acid, (found in leaves and bark of many plant species)

13. Hydrolysable Tannins • They may be hydrolyzed by acids or enzymes such as tannase, (products being sugar and phenolic acids). • Formed by several phenolic acids which are united by ester linkages to a central sugar (glucose) molecule. • Hydrolysable tannins are therefore esters of a sugar and phenolic acid molecules. • The phenolic acid is • in the case of gallotannins – gallic acid • in the case of ellagi-tannins – hexahydroxydiphenic acid (HHDP) and its oxidized derivatives (dehydrohexahydroxydiphenic acid). • Ellagitannins are found in Herb Robert (Geranium species, Meadowsweet and Oak bark).

14. Eg’s of Hydrolysable Tannins Gallotannins: rhubarb, cloves, red rose petals, bearberry leaves, Chinese galls, Turkish galls, hamamelis, chestnut and maple Ellagitannins: pomegranate rind, pomegranate bark, eucalyptus leaves, some Australian kinos, chestnut (Castanea spp), and oak bark.

15. Gallotannins

16. Gallotannins • Gallotannins are the simplesthydrolysabletannins, containing a polyphenolic and a polyol residue. • Polyol: alcohol with multiple hydroxyl groups. • Most of the gallotannins isolated from plants contain a polyol residue derived from D-glucose.

17. Some gallotannin structures

18. Ellagitannins • With more than 500 natural products characterized so far, ellagitannins are the largest group of known tannins. • Ellagitannins are formed from the gallotannins by the oxidative coupling of at least two galloyl units.

19. Condensed Tannins • Are polymers of 2 to 50 (or more) flavonoidunits that are joined by carbon-carbon bonds, which are not susceptible to being cleaved by hydrolysis. • All hydrolyzable tannins and most condensed tannins are water soluble - some very large condensed tannins are insoluble.

20. Condensed Tannins • Condensed tannins are oligomeric and polymeric proanthocyanidins consisting of coupled flavan-3-ol (catechin) units. • (oligomeric or polymeric proanthocyanidins =condensed proanthocyanidins = condensed tannins). • Biosynthetically the condensed tannins are formed by the successive condensation of the single building blocks (up to more than 50 may be included in a single condensed tannin). • The coupling pattern of the catechin units in condensed tannins can vary considerably. NOTE: Oligomeric: a polymer that consists of 2, 3, or 4 monomeres. Polymer: Any of numerous natural and synthetic compounds of usually high molecular weight consisting of up to millions of repeated linked units, each a relatively light and simple molecule.

21. Condensed Tannins/Proanthrocyanidins • They are not readily hydrolysed to simpler molecules and they do not contain a sugar moiety (unlike hydrolysable tannins). • On treatment with acids or enzymes, condensed tannins are converted into red insoluble compounds known as phlobaphenes.

22. Condensed Tannins/Proanthrocyanidins • Phlobaphenes give the characteristic red colour to many drugs such as red cinchona bark, which contains these phlobatannins and their decomposition products. • These tannins are therefore sometimes called catechol tannins (as they yield catechol). • Like catechol itself, their (condensed tannin) solutions turngreenwithferric chloride.

23. Plants rich in Condensed Tannins • Barks: cinnamon, wild cherry, cinchona, willow, acacia (wattle, mimosa), oak bark, Hamamelis. • Flowers: Lime and Hawthorn • Seeds: cocoa, guarana, kola • Fruits: cranberries, grapes, hawthorn • Leaves – Hamamelis, hawthorn, tea leaves (especially green tea) • Extracts and dried juices: catechu, acacia, East Indian kino and eucalyptus kino

24. The structures of the complex tannins are built up from a gallotannin unit or an ellagitanninunit, and a catechin unit. Fig: Acutissimin A, the usual representative of the complextannins. Complex Tannins

25. Tannins - Properties • Water-soluble • Also soluble in dilute alkali, alcohol, glycerol and acetone • Slightly soluble in other organic solvents. • Solutions precipitate heavy metals, alkaloids, glycosides and gelatin.

26. Tannins - Properties • If a dilute ferric chloride solution is added to an aqueous extract of hamamelis leaves (containing both types of tannins), a blue colour is produced which then changes to orange-green as more ferric chloride is added. • Free gallic acid gives an orange colour with potassium iodate.

27. Tannin Properties: Hydrolysable or Condensed? • With ferric salts, gallo-tannins and ellagi-tannins give blue-black precipitates. • Condensed tannins give brown-green precipitates. • Condensed tannins turn red with vanillin and HCL. • Gallo-tannins give a pink colour with potassium iodate. • Ellagitannins react (are coloured) with nitrousacid in the presence of acetic acid. Colour reaction: pink purple  blue. • TLC is used to analyze extracts (visualized under UV light and with the reagents mentioned above) • HPLC can also be used.

28. Some drugs (e.g. tea, Hamamelis leaves and Hamamelis bark) contain both hydrolysable and condensed tannins. Hamamelis leaf & bark

29. Pseudo-Tannins • Pseudo-tannins are compounds of lower molecular weight than true tannins and they do not respond to the goldbeater’s skin test. • Gallic acid: rhubarb • Catechins: catechu, acacia cutch, many Australian kinos, cocoa, guarana and many other drugs containing condensed tannins • Chlorogenic acid: coffee (particularly unroasted) and nux Vormica (a small quantity only). • Ipecacuanhic acid: ipecacuanha

30. Tannins are of wide occurrence in plants and are usually found in greatest quantity in dead or dying cells. They exert an inhibitory effect on many enzymes due to protein precipitation and, hence, may contribute a protective function in barks and heartwoods. Tannin Function

31. Commercial tannins, as used in the leather industry, are obtained from * quebrancho, wattle, and chestnut. Pharmaceutical tannin is prepared from oak galls and yields glucose and gallic acid on hydrolysis. Many commercial samples contain some free gallic acid. Oak galls: pharmaceutical source of tannins Tannin Sources

32. The name quebracho is taken from two Spanish words quebrar and hacha, meaning "to break the axe."  The bark of Quebracho consists of a thick cork layer, traditionally used as a febrifuge.  It is also used to relieve dyspnea (difficult or laboured breathing e.g from emphysema and asthma).  The bark and especially heartwood are used for tanning hides. * Quebrancho Bark

33. Tannin Incompatibilities • Clove and cinnamon contain tannins and also other principal therapeutic constituents. • This may complicate extraction or produce imcompatibilities with other drugs. • E.g. Many alkaloids for example are precipitated by tannins.

34. Tannin Extraction • Tannins are extracted using water and acetone. • Optimal yields are obtained from fresh tissues or freeze-dried tissues. • Optimal yields are not obtained from dried tissues (tannins are irreversibly combined with other polymers)

35. After eliminating the acetone (distillation), the pigments and lipids are removed from the aqueous solution by a solvent extraction. • Ethyl acetate extraction of the aqueous solution separates the dimeric proanthrocyanidins and most gallotannins. • Polymeric proanthrocyanidins and high molecular weight gallotannins remain in the aqueous phase.

36. To obtain pure compounds, the appropriate chromatographic techniques are used (most often one of the gel filtrations) Followed by reverse-phase chromatography (in water and alcohol, or water, alcohol and acetone mixures)

37. Medicinal Properties of Tannins • Therapeutic/medicinal activities result due to the astringency properties. • Tannin containing herbs (drugs) will precipitate proteins. • Externally: waterproof the external layers of the skin and mucosa: Protect underlying layers. • Vasoconstrictor effects – on small superficial vessels

38. Medicinal Properties of Tannins • Limits fluid loss – enhance tissue regeneration in superficial wounds and burns • Internally: Anti-diarrhoeal • Anti-septic effects (anti-bacterial/anti-fungal)  used to Rx infectious diarrhoeas and dermatitis.

39. Anti-oxidant Activity of Tannins • Hydrolysable tannins (more so than condensed tannins) are free-radical scavengers. • Inhibit superoxide ion formation. • Some inhibit lipoxygenase • They do not inhibit cyclo-oxygenase. • Anti-oxidant flavonols and proanthrocyanidins from grape juice and wine are widely considered to be the main principles responsible for the preventative effect of CVD.

40. Enzymatic Inhibition Action • Tannins may act as enzyme inhibitors. • They block 5-lipoxygenase • inhibit angiotensin converting enzyme • hyaluronidase activation • glucosyltransferases (of microorganisms involved in cavity formation).

41. Tannins, including gallo and ellagic acid (epigallitannins), are inhibitors of HIV replication. 1,3,4-Tri-O-galloylquinic acid 3,5-di-O-galloyl-shikimic acid, 3,4,5-tri-O-galloylshikimic acid punicalin Punicalagin inhibited HIV replication in infected H9 lymphocytes with little cytotoxicity. Two compounds, punicalin and punicacortein C, inhibited purified HIV reverse Transcriptase. New (Hydrolysable) Tannin Potential

42. TANNIN CONTAINING HERBS

43. Definition: Turkish galls (Turkey galls, Galla): Are vegetable growths formed on the young twigs of the dyer’s oak, Quercus infectoria (Fagaceae). They result from the decomposition of the eggs of the gall-wasp Adleria gallaetinctoriae. If the galls are not collected and dried, the mature insect bores out of the gall and escapes. Turkish Galls

44. Turkish Galls: Collection Galls are collected by peasants of Turkey and Syria. After collection they are graded according to colour into 3 grades: blue, green and white. These are then found on the London Market. Colour changes occur due to enzymatic secretions of insect.

45. History: Ancient writers recorded the use of an infusion to test for sulphate of iron, possibly the earliest mention of an attempt to detect adulteration by chemical means. Turkish Galls

46. Uses Used as a source of tannic acid. Tannic acid is used as an astringent and styptic. Used for tanning and dyeing. Ink manufacturing. Allied Drugs Chinese and Japanese Galls Hungarian Galls Turkish Galls – Uses & Allied Drugs

47. 50 – 70% tannin – gallotannic acid. Tannic acid: hydrolysable tannin (consisting of gallicacid and glucose). Gallic acid Ellagic acid Sitosterol Starch Calcium oxalate Syringic acid (CNS-active component) Turkish galls - Constituents

48. Definition: Witch hazel leaves consists of the dried leaves of Hamamelis virginiana, Hamamelidaceae). It should contain no less than 30% tannins. Hamamelis Leaf

49. Hamamelis Flowers

50. Witch hazel leaves – Hamamelis Constituents • Gallitannins • Ellagitannins • Free gallic acid • Proanthrocyanidins • Bitter principles • Volatile oil